Targeting the Main Protease of SARS‐CoV‐2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors

The main protease of SARS‐CoV‐2 (Mpro), the causative agent of COVID‐19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed Mpro. Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in‐depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of Mpro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac/Ki=37 500 m−1 s−1, Ki=24.0 nm) and pyridyl ester 17 (kinac/Ki=29 100 m−1 s−1, Ki=10.0 nm), promising drug candidates for further development have been discovered. The main protease (Mpro) of SARS‐CoV‐2 has been recognized as a significant drug target to treat COVID‐19. The design of a new fluorogenic substrate, the recombinant expression of Mpro and the development of an HTS assay were combined with a structure‐based rational approach leading to the discovery of tailored azanitriles and indole pyridyl esters as novel, outstandingly potent Mpro inhibitors.